Short tandem repeats in forensic genetics Flashcards
The history of DNA testing
- RFLP (1985-1990)
- DQ-alpha (1990-1995)
- automated STR (1995-)
what are STRs
- microsatellites
- core repeat motive of 1 to 6 bp
- <350 bp long (alleles)
Where are STRs used?
- human identification
- paternity testing
- missing perso cases
- violent crimes
- mass disasters
How are STRs classified? (4)
- simple repeat
- simple repeat with non-consensus allele
- compound repeat - composed of several elements
- complex repeat
Name characteristics that STRs should possess in order to be used in forensics?
- tetra or penta repeats
- discrete and distinguishable alleles
- amplification of locus should be robust
- a high power of discrimination
- absence of genetic linkage with other loci
- low levels of artifact formation during the amplification
- the ability to be amplified as part of a multiplex PCR
Describe the nomenclature of a STR
D3S1358
D = DNA 3 = number of chromosome S = unique sequence 1358 = particular sequence
What is the amelogenin gene?
- Encodes a protein in tooth enamel
- not an STR, two alleles
- present on both X and Y chromosome
- 6bp longer on Y chromosome
- used for sex determination (but not an absolute indicator)
Describe the workflow of DNA profiling
- DNA extraction
- preparation of PCR reactions
- PCR
- gel electrophoresis
- Sanger sequencing
- Capillary electrophoresis
- analysis with software
- profile
- profile evaluation
How does capillary electrophoresis work?
- glass tube fille with polymer solution
- DNA moves from negative to positive pole (to anode)
- argon laser through capillary, hits label on PCR product and excites fluorophores –> emission of fluorescent light
- light passes through filter (removes background noise)
- light passes through charged coupled device (CCD) camera detecting wavelength
- information to computer –> electropherogram
What is an electropherogram?
A plot of results from an analysis done by electrophoresis - can be used to determine DNA sequence genotypes
Name characteristics of fluorescent based detection assay
- dye binds to 5’ end of primer
- detected real-time during electrophoresis
- up to five different dyes - overlap of loci possible
What is the internal lane size standard?
- fragments of DNA of known length, fluorescently labeled
- size in bp
- allows for comparison and ensures consistency
- comes with the kit
What is the allelic ladder?
- included in kit
- artificial mixture of all human alleles
- reference size for each allele
- migration of PCR products varies
- temperature
- electropheric conditions
Name some complications that can occur with STR analysis
(Also including the profile itself)
- stutter peaks
- split peaks
- pull-ups
- overloaded profiles
- mixed samples
- DNA degradation/low template DNA
- PCR inhibition
What are stutter peaks?
- one repeat unit smaller or larger than the true allele
- caused by strand slippage during extension
- do not interfere with interpretation of profile
- seens as smaller peak next to bigger
- threshold limits: <8-15% of main peak
- shorter STRs more prone to stutter
What are split peaks?
- Taq-polymerase is a terminal transferase - template dependent vs. non-template dependent
- I.e. sometimes it puts a random nucleotide to the extended strand that does not correspond to the template. in 85% A is added
- caused by:
- taq polymerase suboptimal activity
- too much DNA in the PCR
- prevention: add incubation step to PCR
- profile often still readable (only 1bp dif)
What are pull-ups?
- caused by
- matrix file poor quality
- over-amplification
- peaks in profile composed of more than one colour
- easy to recognise: smaller product of same size as allele
What are overloaded profiles and how do you get them?
- overloaded PCR –> CCD camera saturated –> peak height not reliable
- noisy background
- stutterings
- split peaks
- pull-ups
What can occur during low template DNA typing?
- low copy numer <100 pg DNA
- LCN –> increased number of cycles in PCR
- allele drop-in or drop-out
- peak imbalance
- locus drop-out
- increased stutter
What is allelic drop-in?
- false PCR products from unkown DNA –> extra allele
- completely random so rarely repeated
- set up samples many times
- at least two amplifications of same sample as routine
What is allele drop-out?
- one allele preferentially amplified
- caused by
- mutation in primer binding site
- primer mismatch
- degradation
- initial input of DNA too low
- allele size outside normal range goes undetected
- heterozygous appears homozygous
- repeat PCR twice
How to assess peak height imbalance
- peak size is proportional to amout of PCR product
- peak high balance should be 1:1
- cutt of value 60-70%
- height of smaller peak divided by height of larger peak
How does one assess mixed profiles? (5 steps)
- Identify the presence of a mixture
- identify the number of potential contributors
- estimate the relative ratio of the invidividuals contributing to the mixture
- consider all possible genotype combinations
- compare reference samples
STRs vs SNPs
- discrimination power
- occurence
- multiplex capacity
- artifacts
- amount DNA needed
- mixed sample analysis
- phenotypic information
- discrimination power: STRs have the higher one
- occurance:
- STR: every 15kb
- SNPs: every 500bp
- multiplex capacity:
- STR: approx 20
- SNP: approx 50
- artifacts: occur only with STRs
- amount DNA needed:
- STR: 0,5-1ng DNA
- SNP: 100pg DNA (degraded)
- mixed samples:
- STR: possible to analyise
- SNP: difficult to analyse
- phenotypic information: only in SNPs
What does the likelyhood ratio indicate?
It indicates how many times more likely it is that a sample originates from a particular person than that originats from an unrelated person
